I propose here setting up an in vitro system for the study of plasmid RSF1030 replication. I plan to rapidly establish the correspondence of in vivo and in vitro events and to go on to purify and establish the role of new proteins in Escherichia coli and plasmid replication. It is the identification and characterization of host and plasmid proteins involved in replication that is the main thrust of the proposed studies. RSF1030 is a small (5.6 x 10 to the 6th power MW) (drug resistance) plasmid that replicates under relaxed control and requires DNA polymerase I for replication. After developing the in vitro system and characterizing the DNA products synthesized, I plan to work out protocols for dissecting the replication reaction into initiation, elongation, and termination reactions. This will aid in the study of the involvement of the enzymes, termination reactions. This will aid in the study of the involvement of the enzymes, DNA polymerase I, DNA polymerase III, RNA polymerases, ligases, DNA gyrases and other enzymes involved. In addition plasmid replication proteins will be identified and studied. A second aspect of the work will be an investigation of the relationship of RNA synthesis to DNA replication. Ribonucleotides in RSF1030 will be mapped; the mechanism of removal of ribonucleotides will be investigated; enzymes involved will be elucidated.